Lysosomal membrane localization of organic anion transporting polypeptide 2B1 in human hepatocytes.

Abstract

Organic anion transporting polypeptide 2B1 (OATP2B1) is a basolateral plasma membrane uptake transporter expressed in hepatocytes. However, the cellular internalization and degradation pathways of OATP2B1 remain poorly characterized. Filling this knowledge gap is critical for improving predictions of OATP2B1 substrate disposition and OATP2B1-mediated drug interactions. This study investigated the localization and primary degradation pathway of OATP2B1 in human hepatocytes and examined whether OATP2B1 localization was affected by metabolic dysfunction-associated steatohepatitis in human liver tissue. Sandwich-cultured human hepatocytes (SCHHs) were treated with either 25 μM chloroquine (CQ) or 10 μM MG-132 to inhibit lysosomal or proteasomal degradation, respectively. Following treatment, SCHHs were fixed and stained for OATP2B1 and lysosome-associated membrane protein (LAMP) 1, followed by confocal imaging and subsequent object-based quantitative analysis. Lysosomal inhibition by CQ in SCHHs resulted in significant accumulation of intracellular OATP2B1, which displayed major colocalization with the lysosomal marker, LAMP1 (20% ± 9.38% vs 9.5% ± 5.25%; CQ vs control, respectively; P < .0001). In contrast, the OATP2B1 total object volume and intracellular localization remained unchanged after MG-132 treatment. OATP2B1 colocalization with LAMP1 was significantly higher in liver tissue from patients with metabolic dysfunction-associated steatohepatitis (10.47% ± 4.95%) compared with control liver tissue (6.00% ± 2.06%; P < .0001). Super-resolution imaging using stimulated emission depletion microscopy revealed that OATP2B1 was localized on lysosomal membranes. In conclusion, these findings demonstrate that OATP2B1 degrades primarily via lysosomes in human hepatocytes. Lysosomal dysfunction associated with some xenobiotics or disease may increase OATP2B1 localization on lysosomal membranes. SIGNIFICANCE STATEMENT: This study confirms that lysosomes are the predominant degradation pathway for the basolateral membrane protein organic anion transporting polypeptide 2B1 (OATP2B1) in human hepatocytes. Super-resolution imaging revealed that OATP2B1 is localized on lysosomal membranes. Both lysosomal inhibition by chloroquine in vitro and lysosomal dysfunction associated with metabolic dysfunction-associated steatohepatitis in human liver tissue increased OATP2B1 colocalization with the lysosomal membrane marker.